P. aeruginosa persists as a major cause of life-threatening infections for individuals with cystic fibrosis, burns, wounds, cancer, etc. The pili of this bacterium are protein filaments which extend from the ends of the cell. These structures function as virulence factors by serving in adhesion. The long term objective of this project is to determine the role of pilus glycosylation in P. aeruginosa pathogenesis and to ascertain the importance of the glycan in pilus vaccine design. Work from Dr. Castric's laboratory has shown that the pili of strain 1244 are glycosylated. The glycan specificity in these pilin glycosylation reactions is low, a finding which has implications for the pilus vaccine design. The work proposed in this application would explore the extent of this glycan substrate specificity using both in vivo (expressing pilin genes in heterologous strains) and in vitro (glycosylation in cell free extracts) studies. He has also proposed to examine the effect of glycosylation on pilus function, such as adhesion and pilus retraction. Finally, a comprehensive structural analysis of the glycan will be performed, using mass spectrometry and NMR methodologies.